Except for bronchoalveolar lavage, the value of the Gram stain examination of respiratory tract samples for the diagnosis of hospital-acquired pneumonia (HAP) and their potential impact on empiric antibiotic treatment have rarely been assessed. During a 14-mo period, both plugged telescoping catheter (PTC) and endotracheal aspirate (EA) were performed when an HAP was suspected in mechanically ventilated patients. The results of Gram stain examinations and cultures and previous and subsequent antibiotic treatment were prospectively recorded. Two criteria for pneumonia were considered: (1) clinically diagnosed pneumonia, according to attending physicians, based on clinical and radiological evolution and the PTC culture results, and (2) microbiologically proven pneumonia (solely based on the result of quantitative PTC culture). Of 91 episodes of suspected HAP in 51 patients, 27 (30%) episodes were clinically diagnosed as pneumonia. When considering clinically diagnosed pneumonia, the sensitivity and specificity of Gram stain examination were similar (respectively, 89% and 62% for EA and 67% and 95% for PTC). When considering microbiologically proven pneumonia, the sensitivity and specificity of Gram stain examination were, respectively, 91% and 64% for EA and 70% and 96% for PTC. The negative predictive value of Gram stain examination of EA and the positive predictive value of Gram stain examination of PTC were high. Our results suggest that the combination of Gram stain examination of paired PTC and EA may contribute to the early diagnosis of HAP in about two-thirds of mechanically ventilated patients, and guide the empiric therapy when needed. In the remaining one-third of patients, the Gram stain examination is not helpful in predicting HAP.